A3 adenosine receptor antagonists and partial agonists

ABSTRACT

Disclosed are A 3  adenosine receptor antagonists and/or partial agonists of formula (I): wherein R 1  to R 5  are as described herein, as well as pharmaceutical compositions thereof and methods of use thereof. The antagonists or partial agonists find use in treating a number of diseases including cancer, glaucoma, inflammatory diseases, asthma, stroke, myocardial infarction, allergic reactions, rhinitis, poison ivy induced responses, urticaria, scleroderma, arthritis, brain arteriole diameter constriction, bronchoconstriction, and myocardial ischemia, as well as in preventing cardiac ischemia. Also disclosed are radiolabeled compounds of formula (I) and the use thereof in diagnostic imaging of tissues and organs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a U.S. National Phase of International PatentApplication No. PCT/US09/52439, filed Jul. 31, 2009, which claims thebenefit of U.S. Provisional Patent Application No. 61/085,588, filedAug. 1, 2008, the disclosures of the applications are incorporated byreference.

BACKGROUND OF THE INVENTION

There are four subtypes of receptors for adenosine, designated A₁,A_(2A), A_(2B), and A₃. The A₃ adenosine receptor is found primarily inthe central nervous system, brain, testes, and the immune system, whereit appears to be involved in the modulation of release from mast cellsof mediators of the immediate hypersensitivity reaction (Ramkumar etal., J. Biol. Chem., 268, 16887-16890 (1993)).

It is believed that A₃ adenosine receptor selective antagonists shouldserve as cerebroprotective, antiasthmatic, or anti-inflammatory agents.It is also believed that A₃ adenosine receptor selective antagonistsshould serve in the treatment of glaucoma, for example, in reducingintraocular pressure. Research activity is evident in the area of A₃adenosine receptor antagonists; see, for example, U.S. Pat. Nos.6,066,642 and 6,528,516 and WO 2008/055711. Accordingly, there is adesire to find new A₃ adenosine receptor antagonists.

Further, A₃ adenosine receptor partial agonists, are advantageous incardioprotection and produce anti-ischemic effects. Partial agonistsalso tend to have less side effects than full agonists. In addition,partial agonists are less likely to produce desensitization of thereceptor as compared to full agonists. Accordingly, partial agonists canactivate the receptor for a longer duration and achieve longer lastingresponse. Accordingly, there is a desire to find new A₃ adenosinereceptor partial agonists.

BRIEF SUMMARY OF THE INVENTION

The invention provides compounds, pharmaceutical compositions, andmethods of use of the compounds. The compounds of the invention areantagonists, or partial agonists, of the A₃ adenosine receptor and arepurine analogs having substituents at the N⁶-, 2-, and 9-, andoptionally at the 8-position, of the purine core. The compounds have aconstrained ring or a rigid bicyclo[3.1.0]hexane ring at the 9-positionof the purine core, which provides high potency and selectivity to theA₃ adenosine receptor and at the same time lack a substituent on the4′-position of the bicyclo hexane ring. The absence of a 4′-substituentin many of the compounds leads to lack of activation of the A₃ adenosinereceptor. Many of the compounds act as partial agonists.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts a reaction scheme to prepare compounds 7b-13b inaccordance with an embodiment of the invention. a) 7 steps (see Joshi etal. Nucleosides, Nucleotides, and Nucleic Acids 2008, 27, 279 and Joshiet al. J. Org. Chem. 2005, 70, 439); b) TBDPS-Cl, imidazole, DMF; c)NaOH, H₂O, MeOH, reflux; d) 2-mercaptopyridine N-oxide, DCC, toluene; e)(Me₃Si)₃SiH, AIBN, toluene; f) Bu₄NF, THF; g) 2,6-dichloropurine, PPh₃,DIAD, THF; h) RNH₂, EtOH; i) TFA/H₂O/MeOH.

FIG. 2 depicts a reaction scheme to prepare compounds 19a-19g inaccordance with an embodiment of the invention.

FIG. 3 depicts compounds 22a-22g in accordance with an embodiment of theinvention and reaction schemes to prepare compounds 20a-20g and 21a-21g.

FIG. 4 depicts functional antagonism by the compound 7b of the inventionin the guanine nucleotide binding assay ([³⁵S]GTPγS) in membranes of CHOcells expressing human A₃AR.

FIG. 5 depicts functional agonism of compounds 7b and 9b in accordancewith an embodiment of the invention in an assay of adenylate cyclasemembranes of CHO cells expressing hA₃AR. The full agonist NECA(5′-N-ethylcarboxamidoadenosine), representing 100% efficiency, is showncomparison.

FIG. 6 depicts a reaction scheme for the preparation and radioiodinationof compound 7b.

FIG. 7A depicts the non-specific, specific, and total binding of [¹²⁵I]7b on mouse A₃ adenosine receptor. FIG. 7B depicts the extent ofspecific binding as a function of the concentration of the compound.

FIG. 8 depicts the biodistribution of Br-76 labeled compound 9b at 15,60, and 120 min post injection in rats. The Y-axis represents % InitialDose per gram and X-axis shows various organs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on the concept that compounds havinga ring constrained substituent or a rigid bicyclo[3.1.0]hexane ring atthe 9-position which provides high potency as an antagonist andselectivity to the A₃ adenosine receptor, or as a partial agonist of theA₃ adenosine receptor, and at the same time lack a substituent on the4-position of the bicycle hexane ring.

Accordingly, the present invention provides a compound of Formula I:

wherein

R¹ is selected from the group consisting of hydrogen, C₁-C₆ alkyl, C₁-C₆alkoxy, hydroxyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl C₁-C₆ alkyl, C₃-C₈dicycloalkyl C₁-C₆ alkyl, C₇-C₁₂ bicycloalkyl C₁-C₆ alkyl, C₇-C₁₄tricycloalkyl C₁-C₆ alkyl, C₆-C₁₄ aryl, C₆-C₁₄ aryl C₁-C₆ alkyl, C₆-C₁₄diaryl C₁-C₆ alkyl, C₆-C₁₄ aryl C₁-C₆ alkoxy, C₁-C₆ alkyl carbonyl,sulfonyl, C₁-C₆ alkyl sulfonyl, C₆-C₁₄ aryl sulfonyl, heterocyclyl C₁-C₆alkyl, heterocyclyl, heteroaryl C₁-C₆ alkyl, 4-[[[4-[[[(2-amino C₁-C₆alkyl)amino]-carbonyl]-C₁-C₆ alkyl]aniline]carbonyl]C₁-C₆ alkyl]C₆-C₁₄aryl, and C₆-C₁₄ aryl C₃-C₈ cycloalkyl, wherein the aryl or heterocyclylportion of R¹ is optionally substituted with one or more substituentsselected from the group consisting of halo, amino, hydroxyl, carboxy,C₁-C₆ alkoxycarbonyl, aminocarbonyl, C₁-C₆ alkylaminocarbonyl, C₁-C₆dialkyl aminocarbonyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxy, C₆-C₁₄ aryloxy, hydroxy C₁-C₆ alkyl, hydroxy C₂-C₆ alkenyl,hydroxy C₂-C₆ alkynyl, carboxy C₁-C₆ alkyl, carboxy C₂-C₆ alkenyl,carboxy C₂-C₆ alkynyl, aminocarbonyl C₁-C₆ alkyl, aminocarbonyl C₂-C₆alkenyl, aminocarbonyl C₂-C₆ alkynyl, and C≡C—(CH₂)_(n)—COR⁷ wherein R⁷is selected from the group consisting of OH, OR⁸, and NR⁹R¹⁰, wherein R⁸is selected from the group consisting of C₁-C₆ alkyl, C₃-C₈ cycloalkyl,C₃-C₈ cycloalkyl C₁-C₆ alkyl, C₃-C₈ dicycloalkyl C₁-C₆ alkyl, C₇-C₁₂bicycloalkyl C₁-C₆ alkyl, C₇-C₁₄ tricycloalkyl C₁-C₆ alkyl, C₆-C₁₄ aryl,C₆-C₁₄ aryl C₁-C₆ alkyl, C₆-C₁₄ and diaryl C₁-C₆ alkyl; and R⁹ and R¹⁰are independently selected from the group consisting of hydrogen, C₁-C₆alkyl, and (CH₂)_(n)R¹¹ wherein R¹¹ is NR¹²R¹³, wherein R¹² and R¹³ areindependently selected from the group consisting of hydrogen, C₁-C₆alkyl, and COR¹⁴ wherein R¹⁴ is hydrogen or C₁-C₆ alkyl; wherein n is aninteger from 1 to 10; and the alkyl or cycloalkyl portion of R¹ isoptionally substituted with one or more substituents selected from thegroup consisting of halo, amino, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₆-C₁₄aryloxy, C₁-C₆ hydroxyalkyl, C₂-C₆ hydroxyalkenyl, C₂-C₆ hydroxyalkynyl, aminocarbonyl C₁-C₆ alkoxy, and C₆-C₁₄ aryl C₁-C₆ alkoxy;

R² is selected from the group consisting of hydrogen, halo, amino,hydrazido, mercapto, C₁-C₂₀ alkylamino, C₆-C₁₄ aryl amino, C₆-C₁₄aryloxy, C₁-C₂₀ alkyl, C₁-C₂₀ alkoxy, C₁-C₂₀ thioalkoxy, pyridylthio,C₇-C₁₂ cycloalkyl C₁-C₂₀ alkyl, C₇-C₁₂ bicycloalkyl C₁-C₂₀ alkyl, C₇-C₁₂bicycloalkenyl C₁-C₂₀ alkyl, C₆-C₁₄ aryl C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl,C₇-C₁₂ cycloalkyl C₂-C₂₀ alkenyl, C₇-C₁₂ bicycloalkyl C₂-C₂₀ alkenyl,C₇-C₁₂ bicycloalkenyl C₂-C₂₀ alkenyl, C₆-C₁₄ aryl C₂-C₂₀ alkenyl, C₂-C₂₀alkynyl, —C≡C—(CH₂)_(m)—C(═O)—O—C₁-C₆ alkyl,—C≡C—(CH₂)_(m)—C(═O)—NH—(CH₂)_(n)—NH₂, —C≡C—(CH₂)_(m)—C₁-C₆ alkyl,—C≡C—(CH₂)_(m)-aryl, wherein m and n are independently 1 to 10, C₇-C₁₂cycloalkyl C₂-C₂₀ alkynyl, C₇-C₁₂ bicycloalkyl C₂-C₂₀ alkynyl, C₇-C₁₂bicycloalkenyl C₂-C₂₀ alkynyl, C₆-C₁₄ aryl C₂-C₂₀ alkynyl, and thealkyl, cycloalkyl, or aryl portion of R² is optionally substituted withone or more substituents selected from the group consisting of halo,hydroxyl, amino, alkylamino, dialkylamino, sulfur, carboxy,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, aminoalkyl aminocarbonyl, and trialkylsilyl;

R³ and R⁴ are independently selected from the group consisting ofhydroxyl, amino, thiol, ureido, C₁-C₆ alkyl carbonylamino, hydroxy C₁-C₆alkyl, and hydrazinyl; and

R⁵ is selected from the group consisting of hydrogen, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, heteroaryl, and C₁-C₆ aminoalkyl;

or a pharmaceutically acceptable salt thereof.

The term “aryl” refers to aromatic moieties such as phenyl, naphthyl,anthracenyl, and biphenyl. The term “heterocyclyl” refers to 3-7membered rings which can be saturated or unsaturated or heteroaromatic,comprising carbon and one or more heteroatoms such as O, N, and S, andoptionally hydrogen; optionally in combination with one or more aromaticrings. Examples of heterocyclyl groups include pyridyl, piperidinyl,piperazinyl, pyrazinyl, pyrolyl, pyranyl, tetrahydropyranyl,tetrahydrothiopyranyl, pyrrolidinyl, furanyl, tetrahydrofuranyl,thienyl, furyl, thiophenyl, tetrahydrothiophenyl, purinyl, pyrimidinyl,thiazolyl, thiazolidinyl, thiazolinyl, oxazolyl, tetrazolyl, tetrazinyl,benzoxazolyl, morpholinyl, thiomorpholinyl, quinolinyl, andisoquinolinyl. Examples of heteroaryl alkyl include heteroaryl methylsuch as 2- or 3-methyl substituted groups, e.g., thienylmethyl,pyridylmethyl, and furylmethyl.

The alkyl, alkoxy, and alkylamino groups can be linear or branched. Whenan aryl group is substituted with a substituent, e.g., halo, amino,alkyl, hydroxyl, alkoxy, and others, the aromatic ring hydrogen isreplaced with the substituent and this can take place in any of theavailable hydrogens, e.g., 2, 3, 4, 5, and/or 6-position wherein the1-position is the point of attachment of the aryl group in the compoundof the present invention.

The term “halo” refers to fluorine, chlorine, bromine, and iodine.

Examples of bicycloalkyls include norbornyl, s-endonorbornyl,carbamethylcylopentyl, and bicyclohexyl. An example of a tricycloalkylis adamantyl.

The phrase “salt” or “pharmaceutically acceptable salt” is intended toinclude nontoxic salts synthesized from the parent compound whichcontains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with a stoichiometric amount of the appropriatebase or acid in water or in an organic solvent, or in a mixture of thetwo. Generally, nonaqueous media such as ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 18th ed., Mack PublishingCompany, Easton, Pa., 1990, p. 1445, and Journal of PharmaceuticalScience, 66, 2-19 (1977). For example, they can be a salt of an alkalimetal (e.g., sodium or potassium), alkaline earth metal (e.g., calcium),or ammonium of salt.

Examples of pharmaceutically acceptable salts for use in the presentinventive pharmaceutical composition include those derived from mineralacids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric,nitric and sulphuric acids, and organic acids, such as tartaric, acetic,citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic,maleic and arylsulfonic, for example, benzenesulfonic andp-toluenesulfonic, acids.

In accordance with an embodiment of the invention, R¹ is selected fromthe group consisting of C₆-C₁₄ aryl C₁-C₆ alkyl and C₆-C₁₄ aryl C₃-C₈cycloalkyl, wherein the aryl portion of R¹ is optionally substitutedwith one or more substituents selected from the group consisting ofhalo, amino, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₆-C₁₄ aryloxy, hydroxy C₁-C₆alkyl, hydroxy C₂-C₆ alkenyl, hydroxy C₂-C₆ alkynyl, aminocarbonyl C₁-C₆alkoxy, and C₆-C₁₄ aryl C₁-C₆ alkoxy; and in a particular embodiment, R¹is selected from the group consisting of benzyl, phenyl cyclopropyl, or1-naphthyl methyl, wherein the phenyl or naphthyl portion of R¹ isoptionally substituted with one or more substituents selected from thegroup consisting of halo, amino, hydroxyl, carboxy, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkyl aminocarbonyl, C₁-C₆ alkyl,C₁-C₆ alkoxy, phenoxy, hydroxy C₁-C₆ alkyl, hydroxy C₂-C₆ alkenyl, andhydroxy C₂-C₆ alkynyl.

In a specific embodiment of the invention, R¹ is benzyl, phenylcyclopropyl, or 1-naphthyl methyl, wherein the phenyl or naphthylportion of R¹ is optionally substituted with one or more substituentsselected from the group consisting of halo, hydroxyl, and alkoxy.Examples of R¹ are benzyl and benzyl substituted with one or moresubstituents selected from the group consisting of halo and C₁-C₆alkoxy.

In any of the embodiments above, R¹ is selected from the groupconsisting of 3-chlorobenzyl, 3-bromobenzyl, 3-iodobenzyl,2-hydroxy-5-methoxy-benzyl, and 2,5-dimethoxybenzyl. In an embodiment,the phenyl cyclopropyl is trans-2-phenyl-1-cyclopropyl.

In any of the embodiments above, R² is halo, specifically chloro, bromo,or iodo, or R² is —C≡C—(CH₂)_(m)—CH₃, —C≡C—(CH₂)_(m)-aryl,—C≡C—(CH₂)_(m)—C(═O)—O—CH₃, —C≡C—(CH₂)_(m)-—C(═O)—NH—(CH₂)_(n)—NH₂,wherein m and n are independently 1 to 10, where in certain embodimentsm and n are 2 to 6, and in certain other embodiments m and n are 3 to 5,and wherein the CH₃ or aryl group is optionally substituted with one ormore substituents selected from the group consisting of halo, hydroxyl,amino, alkylamino, dialkylamino, sulfur, carboxy, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkyl aminocarbonyl, aminoalkylaminocarbonyl, and trialkylsilyl; or a pharmaceutically acceptable saltthereof.

In any of the embodiments above, R³ and R⁴ are particularly hydroxyl.

In any of the embodiments above, R⁵ is particularly hydrogen.

The term “one or more substituents” in any of the embodiments of theinvention refers to 1, 2, 3, 4, or more substituents.

Particular examples of compounds of the invention are those wherein R²is chloro, R¹ is 3-chlorobenzyl, 3-iodobenzyl, 3-bromobenzyl,1-naphthylmethyl, 2,5-dimethoxy-benzyl, 2-hydroxy-5-methoxybenzyl, ortrans-2-phenyl-cyclopropyl, R³ and R⁴ are hydroxyl, and R⁵ is hydrogen.

Many of the compounds described above have antagonistic as well aspartial agonistic properties at the A₃ adenosine receptor, dependingupon the parameter studied. The definition of antagonist or agonist ishighly dependent upon the cell system and the parameter studied,receptor density, species, and the like.

The compounds of the invention can be prepared by any suitable method.For example, FIG. 1 illustrates a method of preparing compounds 7b-13b.FIG. 2 illustrates a method of preparing compounds 19a-19g. FIG. 3illustrates a method of preparing compounds 20a-20g and 21a-21g.

The present invention further provides a pharmaceutical compositioncomprising a compound as described above and a pharmaceuticallyacceptable carrier. The present invention provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and aneffective amount, e.g., a therapeutically effective amount, including aprophylactically effective amount, of one or more of the aforesaidcompounds, or salts thereof, of the present invention.

The pharmaceutically acceptable carrier can be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with thecompound, and by the route of administration. It will be appreciated byone of skill in the art that, in addition to the following describedpharmaceutical compositions; the compounds of the present invention canbe formulated as inclusion complexes, such as cyclodextrin inclusioncomplexes, or liposomes.

The pharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, or diluents, are well known to thosewho are skilled in the art and are readily available to the public. Itis preferred that the pharmaceutically acceptable carrier be one whichis chemically inert to the active compounds and one which has nodetrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularactive agent, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of the pharmaceutical composition of the present invention.The following formulations for oral, aerosol, parenteral, subcutaneous,intravenous, intraarterial, intramuscular, interperitoneal, intrathecal,rectal, and vaginal administration are merely exemplary and are in noway limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations may include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, and the polyethylene alcohols, eitherwith or without the addition of a pharmaceutically acceptablesurfactant, suspending agent, or emulsifying agent. Capsule forms can beof the ordinary hard- or soft-shelled gelatin type containing, forexample, surfactants, lubricants, and inert fillers, such as lactose,sucrose, calcium phosphate, and cornstarch. Tablet forms can include oneor more of lactose, sucrose, mannitol, corn starch, potato starch,alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, calcium stearate, zinc stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, disintegratingagents, moistening agents, preservatives, flavoring agents, andpharmacologically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin, or sucrose and acacia, emulsions, gels,and the like containing, in addition to the active ingredient, suchcarriers as are known in the art.

The compounds of the present invention, alone or in combination withother suitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The compound can be administered in a physiologically acceptable diluentin a pharmaceutical carrier, such as a sterile liquid or mixture ofliquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol, or hexadecylalcohol, glycols, such as propylene glycol or polyethylene glycol,glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers,such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acidester or glyceride, or an acetylated fatty acid glyceride with orwithout the addition of a pharmaceutically acceptable surfactant, suchas a soap or a detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters. Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include (a)cationic detergents such as, for example, dimethyl dialkyl ammoniumhalides, and alkyl pyridinium halides, (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylene-polypropylene copolymers, (d) amphoteric detergents suchas, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazolinequaternary ammonium salts, and (3) mixtures thereof.

The parenteral formulations will typically contain from about 0.5 toabout 25% by weight of the active ingredient in solution. Suitablepreservatives and buffers can be used in such formulations. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5 to about15% by weight. Suitable surfactants include polyethylene sorbitan fattyacid esters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The compounds of the present invention may be made into injectableformulations. The requirements for effective pharmaceutical carriers forinjectable compositions are well known to those of ordinary skill in theart. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co.,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986).

Additionally, the compounds of the present invention may be made intosuppositories by mixing with a variety of bases, such as emulsifyingbases or water-soluble bases. Formulations suitable for vaginaladministration may be presented as pessaries, tampons, creams, gels,pastes, foams, or spray formulas containing, in addition to the activeingredient, such carriers as are known in the art to be appropriate.

The present invention also provides a method of treating a disease in ananimal, e.g., a mammal, comprising administering to the animal aneffective amount of a compound or a pharmaceutically acceptable salt ofthe invention, wherein the disease is selected from the group consistingof cancer, glaucoma, inflammatory diseases, asthma, stroke, myocardialinfarction, allergic reactions, rhinitis, poison ivy induced responses,urticaria, scleroderma, arthritis, brain arteriole diameterconstriction, bronchoconstriction, and myocardial ischemia. Theinvention also provides a method for selectively inactivating an A₃adenosine receptor, or partially activating an A₃ adenosine receptor, inas animal in need thereof, comprising administering to the mammal aneffective amount of a compound or pharmaceutically acceptable salt ofthe invention. The methods of the invention can be applied to anysuitable mammal, particularly human.

The term “animal” refers to any member of the animal kingdom. Inembodiments, “animal” refers to a human at any stage of development. Inembodiments, “animal” includes mammals, birds, reptiles, amphibians,fish, and worms. In certain embodiments, the non-human animal is amammal, e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, acat, a sheep, cattle, a primate, or a pig. The animal may also be atransgenic animal, genetically engineered animal, or a clone.

The present invention further provides a method for inactivating A₃adenosine receptors, or partially activating such a receptor, in a cellcomprising contacting the cell with an effective amount of one or moreof the inventive compounds or a pharmaceutically acceptable saltthereof. The contacting can be in vitro or in vivo. When the contactingis done in vitro, the contacting can be done by any suitable method,many of which are known in the art. For example, the cell can beprovided in a culture medium and the inventive compound introduced intothe culture medium per se, or as a solution of the compound in anappropriate solvent.

The present invention further provides a method of cardioprotection forpreventing or reducing ischemic damage to the heart in an animal in needthereof comprising administering to the animal a compound or salt asdescribed above, particularly, a compound or salt of formula I, whereinR¹ is 3-bromobenzyl or 3-iodobenzyl, R² is halo, R³ and R⁴ are hydroxyl,and R⁵ is hydrogen.

The compounds or salts thereof can be used in any suitable dose.Suitable doses and dosage regimens can be determined by conventionalrange finding techniques. Generally treatment is initiated with smallerdosages, which are less than the optimum dose. Thereafter, the dosage isincreased by small increments until optimum effect under thecircumstances is reached. For convenience, the total daily dosage may bedivided and administered in portions during the day if desired. Inproper doses and with suitable administration of certain compounds, thepresent invention provides for a wide range of responses. Typically thedosages range from about 0.001 to about 1000 mg/kg body weight of theanimal being treated/day. For example, in embodiments, the compounds orsalts may be administered from about 100 mg/kg to about 300 mg/kg, fromabout 120 mg/kg to about 280 mg/kg, from about 140 mg/kg to about 260mg/kg, from about 150 mg/kg to about 250 mg/kg, from about 160 mg/kg toabout 240 mg/kg, of subject body weight per day, one or more times aday, to obtain the desired therapeutic effect.

In accordance with another embodiment, the invention providesisotopically labeled compounds described above, for example, compoundslabeled with a radioactive or non-radioactive isotope, for use in thedetermination of drug/tissue distribution assays, in the manipulation ofoxidative metabolism via the primary kinetic isotope effect, inidentifying potential therapeutic agents for the treatment of diseasesor conditions associated with target-receptor mediation. The compoundsof the invention can be prepared with a radioactive isotope. Anysuitable atom can be replaced with a radioactive isotope, for example, acarbon atom, hydrogen atom, a halogen atom, a sulfur atom, nitrogenatom, or an oxygen atom can be replaced with a corresponding isotope.Thus, for example, a halogen atom can be replaced with ¹⁸F, ³⁶Cl, ⁷⁶Br,⁷⁷Br, ⁸²Br, ¹²²I, ¹²³I, ¹²⁵I, or ¹³¹I. The use of radiolabeled compoundsthat may be detected using imaging techniques, such as the Single PhotonEmission Computerized Tomography (SPECT), Magnetic ResonanceSpectroscopy (MRS), or the Positron Emission Tomography (PET), are knownin the art. See, for example, U.S. Pat. Nos. 6,395,742 and 6,472,667.

In accordance with a further embodiment, the invention provides aradiolabeled compound of Formula I:

wherein

R¹ is selected from the group consisting of C₆-C₁₄ aryl, C₆-C₁₄ arylC₁-C₆ alkyl, C₆-C₁₄ diaryl C₁-C₆ alkyl, C₆-C₁₄ aryl C₁-C₆ alkoxy, C₆-C₁₄aryl sulfonyl, heterocyclyl C₁-C₆ alkyl, heterocyclyl, heteroaryl C₁-C₆alkyl, 4-[[[4-[[[(2-amino C₁-C₆ alkyl)amino]-carbonyl]-C₁-C₆alkyl]aniline]carbonyl]C₁-C₆ alkyl]C₆-C₁₄ aryl, and C₆-C₁₄ aryl C₃-C₈cycloalkyl, wherein the aryl or heterocyclyl portion of R¹ issubstituted with one or more halogen atoms that are radioactive;

R² is selected from the group consisting of hydrogen, halo, amino,hydrazido, mercapto, C₁-C₂₀ alkylamino, C₆-C₁₄ aryl amino, C₆-C₁₄aryloxy, C₁-C₂₀ alkyl, C₁-C₂₀ alkoxy, C₁-C₂₀ thioalkoxy, pyridylthio,C₇-C₁₂ cycloalkyl C₁-C₂₀ alkyl, C₇-C₁₂ bicycloalkyl C₁-C₂₀ alkyl, C₇-C₁₂bicycloalkenyl C₁-C₂₀ alkyl, C₆-C₁₄ aryl C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl,C₇-C₁₂ cycloalkyl C₂-C₂₀ alkenyl, C₇-C₁₂ bicycloalkyl C₂-C₂₀ alkenyl,C₇-C₁₂ bicycloalkenyl C₂-C₂₀ alkenyl, C₆-C₁₄ aryl C₂-C₂₀ alkenyl, C₂-C₂₀alkynyl, carboxy alkyl C₂-C₂₀ alkynyl, —C≡C—(CH₂)_(m)—C(═O)—O—C₁-C₆alkyl, —C≡C—(CH₂)_(m)—C(═O)—NH—(CH₂)_(n)—NH₂, —C≡C—(CH₂)_(m)—C₁-C₆alkyl, —C≡C—(CH₂)_(m)-aryl, wherein m and n are independently 1 to 10,C₇-C₁₂ cycloalkyl C₂-C₂₀ alkynyl, C₇-C₁₂ bicycloalkyl C₂-C₂₀ alkynyl,C₇-C₁₂ bicycloalkenyl C₂-C₂₀ alkynyl, C₆-C₁₄ aryl C₂-C₂₀ alkynyl, andthe alkyl, cycloalkyl, or aryl portion of R² is optionally substitutedwith one or more substituents selected from the group consisting ofhalo, hydroxyl, amino, alkylamino, dialkylamino, sulfur, carboxy,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, aminoalkyl aminocarbonyl, and trialkylsilyl;

R³ and R⁴ are independently selected from the group consisting ofhydroxyl, amino, thiol, ureido, C₁-C₆ alkyl carbonylamino, hydroxy C₁-C₆alkyl, and hydrazinyl; and

R⁵ is selected from the group consisting of hydrogen, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, heteroaryl, and C₁-C₆ aminoalkyl;

or a pharmaceutically acceptable salt thereof.

The halogen atom of the radiolabeled compound or salt in R¹ of theinvention can be any suitable isotope, for example, ¹⁸F, ⁷⁶Br, ¹²⁵I,preferably ⁷⁶Br or ¹²⁵I.

In a particular embodiment, the invention provides radiolabeledcompounds or salts wherein R¹ is 3-bromobenzyl or 3-iodobenzyl, R² ishalo, R³ and R⁴ are hydroxyl, and R⁵ is hydrogen.

Accordingly, the present invention further provides a method ofdiagnostic imaging of an A₃ adenosine receptor in a tissue or organ ofan animal comprising administering an effective amount of a radiolabeledcompound or salt as described above to the animal and obtaining an imageof the organ or tissue of the animal. The image can be obtained by anysuitable imaging technique, for example, SPECT, MRS, and/or PET.

The present invention also provides a diagnostic method for determininga treatment of a patient for a possible agonist or antagonist of the A₃adenosine receptors, the treatment comprising:

(a) administering a radiolabeled compound or salt as described above;

(b) obtaining a biological sample from the patient;

(c) determining the level of expression of the A₃ adenosine receptor;

(d) comparing the level of expression of the receptor to that of anormal population; and

(e) if the patient's level of expression is higher than that of thenormal population, determining a treatment regimen comprisingadministering an agonist or antagonist of the adenosine receptor whoseexpression was higher in the patient than that of the normal population.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates a method of preparing compounds in accordancewith an embodiment of the invention. D-ribose was protected withTBDPS-Cl followed by alkaline hydrolysis, thus providing acid 2.Reductive decarboxylation of acid 2 was carried out using non-toxictris(trimethylsilyl)silane as a hydrogen donor and produced the silylether 3 in 40% yield. The silyl ether 3 was deprotected with TBAF. Theresultant alcohol 4 was converted into a key dichloropurine derivative 6through a Mitsonobu reaction (FIG. 1). Derivative 6 reacted with anexcess of the corresponding primary amine to give the N⁶ substituted and2′,3′-isopropylidene protected derivatives compounds 7a-13a, followed byacid catalyzed deprotection to give the N⁶-3-halobenzyl and relatedarylmethyl derivatives 7b-13b.

(1R,2S,3R,4R,5R)-3,4-O-(Isopropylidene)-2-O-(tert-butyldiphenylsilyl)-2,3,4-trihydroxybicyclo[3.1.0]hexane-1-carboxylicacid (2)

tert-Butyldiphenylsilyl chloride (2.70 g, 10 mmol) and triethylamine(2.0 g, 20 mmol) were added to a solution of alcohol 1 (prepared fromD-ribose following the standard procedure (Joshi et al. supra) 1.22 g, 5mmol) and imidazole (140 mg, 2 mmol) in DMF (3 mL) while stirring atroom temperature. The solution was stirred at 60° C. for 16 h. Thereaction mixture was cooled to room temperature and diluted with a 4:1ethyl acetate-hexane mixture (50 mL), washed with water, dried, andsolvent was evaporated. The residue was purified by flash chromatography(0 to 10% ethyl-acetate-hexane) to give ethyl(1R,2S,3R,4R,5R)-2,3-O-(isopropylidene)-4-O-(tert-butyldiphenylsilyl)-2,3,4-trihydroxybicyclo[3.1.0]hexane-1-carboxylate.The compound was dissolved in MeOH (5 mL), 2N aq. NaOH (5 mL) was added,and the reaction mixture was refluxed for 2 h. The reaction mixture wasneutralized with NaH₂PO₄, and extracted with DCM. The combined DCMsolutions were dried and evaporated, and the residue was purified byflash chromatography to give title compound 2 (1.65 g, 73%). ¹H NMR(CDCl₃), δ: 7.72 (d, 4H, J=7.8 Hz), 7.39 (m, 6H), 5.05 (d, 1H, J=6.3Hz), 4.43 (t, 1H, J=6.0 Hz), 4.08 (t, 1H, J=6.6 Hz), 2.26 (m, 1H), 1.97(s, 3H), 1.56 (s, 3H), 1.52 (m, 1H), 1.21 (s, 3H), 1.08 (s, 9H).

(1S,2S,3R,4R,5R)-3,4-O-(Isopropylidene)-2-O-(tert-butyldiphenylsilyl)-2,3,4-trihydroxybicyclo[3.1.0]hexane(3)

A 1M solution of DCC in oxygen-free toluene (0.96 mL) was added to asolution of acid 2 (363 mg, 0.80 mmol), 2-mercaptopyridine N-oxide (112mg, 0.88 mmol), and AIBN (40 mg, 0.24 mmol) in dry oxygen-free toluene(4 mL). The reaction mixture was stirred for 4 h at 25° C.,tris(trimethylsilyl)silane (0.50 mL, 1.6 mmol) was added, and thereaction mixture was heated at 85° C. for 4 h. The reaction mixture wasevaporated, and the residue was separated by flash chromatography (0 to10% ethyl acetate-hexane mixture) to afford the title compound 3 (121mg, 40%). ¹H NMR (CDCl₃), δ: 7.76 (d, 4H, J=7.8 Hz), 7.39 (m, 6H), 4.66(t, 1H, J=6.0 Hz), 4.44 (t, 1H, J=6.6 Hz), 4.03 (t, 1H, J=6.6 Hz), 1.6(m, 1H), 1.57 (s, 3H), 1.45 (m, 1H), 1.33 (s, 1H), 1.20 (s, 3H), 1.09(s, 9H), 0.58 (m, 1H).

(1R,2R,3S,4S,5S)-2,3-O-(Isopropylidene)-2,3,4-trihydroxy-bicyclo[3.1.0]hexane(4), Method B

A 1M solution of tert-butylammonium fluoride in THF (1 mL) was added toa solution of silylether 3 (102 mg, 0.25 mmol) in THF (1 mL). Thereaction mixture was left at 20° C. for 16 h and evaporated. The residuewas diluted with ethyl acetate (20 mL) and washed with a small amount ofbrine. The ethyl acetate solution was dried and evaporated, and theresidue was purified by flash chromatography to afford the titlecompound 4 (33 mg, 84%). ¹H NMR and MS are provided under Method A.

General Procedure for Preparation of Compounds 7b-13b

An amine (RNH₂ in Scheme 3, 0.5 mmol) was added to a solution of 6 (20mg, 0.06 mmol) in DCM (0.1 mL). The reaction mixture was stirred at roomtemperature for 16 h. The solvent was removed under vacuum, and theresidue was separated by flash chromatography (30 to 100% ethylacetate-hexane) to afford the corresponding 6-alkylaminopurinederivative that was dissolved in a mixture of MeOH (4 mL), TFA (0.2 mL)and water (2 mL). The reaction mixture was stirred at 70° C. for 16 h,and then evaporated. The residue was evaporated twice with water, andthe residue was purified by flash chromatography (50 to 100% ethylacetate).

(1′R,2′R,3′S,4′R,5′S)-4′-[2-Chloro-6-(3-iodobenzylamino)purine]-2′,3′-dihydroxybicyclo-[3.1.0]hexane(7b)

Yield 15 mg (51% ¹H NMR (CD₃OD), δ: 8.16 (s, 1H), 7.49 (s, 1H), 7.60 (d,1H, 8.5 Hz), 7.40 (d, 1H, 8.5 Hz), 7.10 (t, 1H, 8.5 Hz), 4.71 (s, 2H),3.90 (d, 3.3 Hz, 1H), 3.65 (s, 1H), 2.05-1.95 (m, 1H), 1.67-1.63 (m,1H), 1.36 (s, 1H), 1.31-1.27 (m, 1H), 0.95-0.87 (m, 1H), 0.77-0.75 (m,1H). HRMS calculated for C₁₈H₁₈ClIN₅O₂ ⁺(M+H)⁺: 498.0194. found,498.0194. HPLC: RT 21.6 min (98%) in solvent system A, 17.0 min (98%) insystem B.

(1′R,2′R,3′S,4′R,5′S)-4′-[2-Chloro-6-(3-chlorobenzylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(8b)

Yield 58%. ¹H NMR (CD₃OD), δ: 8.16 (br. s., 1H), 7.41 (s, 1H), 7.29 (m,3H), 4.79 (s, 1H), 4.75 (br. s, 2H), 4.70 (br. t., 1H, J=5.4 Hz), 3.86(d, 1H, J=6.6 Hz), 1.97 (m, 1H), 1.65 (m, 1H), 1.30 (m, 1H), 0.75 (m,1H). HRMS (ESI MS m/z): calculated for C₁₈H₁₈Cl₂N₅O₂ ⁺(M+H)⁺, 406.0832.found, 406.0825. HPLC RT 20.3 min (98%) in solvent system A, 15.6 min(98%) in system B.

(1′R,2′R,3′S,4′R,5′S)-4′-[2-Chloro-6-(3-bromobenzylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(9b)

Yield 65%. ¹H NMR (CD₃OD): 8.03 (s, 1H), 7.45 (s, 1H), 7.29 (m, 2H),7.12 (t, 1H, J=7.8 Hz), 4.68 (s, 1H), 4.63 (br. s, 2H), 4.59 (br. t.,1H, J=5.4 Hz), 3.79 (d, 1H, J=6.6 Hz), 1.86 (m, 1H), 1.55 (m, 1H), 1.20(m, 1H), 0.64 (m, 1H). HRMS (ESI MS m/z) calculated for C₁₈H₁₈BrClN₅O₂ ⁺(M+H)⁺, 450.0327. found 450.0315. HPLC RT 20.74 min (98%) in solventsystem A, 16.1 min (99%) in system B.

(1′R,2′R,3′S,4′R,5′S)-4′-[2-Chloro-6-(1-naphthylamino)purine]-2′,3′-O-dihydroxybicyclo[3.1.0]hexane(10b)

Yield 48%. ¹H NMR (CD₃OD): 8.13 (br. d., 2H, J=7.8 Hz), 7.84 (m, 2H),7.49 (m, 4H), 5.21 (s, 1H), 4.79 (br. s, 1H), 4.78 (br. s, 2H), 4.67(br. t., 1H, J=5.1 Hz), 3.88 (d, 1H, J=6.6 Hz), 1.93 (m, 1H), 1.62 (m,1H), 1.25 (m, 1H), 0.73 (m, 1H). HRMS (ESI MS m/z) calculated forC₂₂H₂₁ClN₅O₂ ⁺ (M+H)⁺, 422.1378. found 422.1385. HPLC RT 21.5 min (97%)in solvent system A, 17.0 min (98%) in system B.

(1′R,2′R,3′S,4′R,5′S)-4′-[2-Chloro-6-(2,5-dimethoxybenzylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(11b)

Yield 44%. ¹H NMR (CD₃OD): 8.4 (very br. s, 1H), 6.95 (s, 1H, J=2.7 Hz),6.89 (d, 1H, J=9.3 Hz), 6.78 (dd, 1H, J=2.7, 9.0 Hz), 4.80 (s, 1H), 4.75(br. m, 3H), 3.87 (d, 1H, J=6.3 Hz), 3.83 (s, 3H), 3.71 (s, 3H), 1.95(m, 1H), 1.64 (m, 1H), 1.29 (m, 1H), 0.74 (m, 1H). HRMS (ESI MS m/z)calculated for C₂₀H₂₃ClN₅O₄ ⁺ (M+H)⁺, 432.1433. found 432.1439. HPLC RT18.7 min (98%) in solvent system A, 16.6 min (98%) in system B.

(1′R,2′R,3′S,4′R,5′S)-4′-[2-Chloro-6-(2-hydroxy-5-methoxybenzylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(12b)

Yield 39%. ¹H NMR (CD₃OD): 8.07 (s, 1H), 6.60-6.82 (m, 3H), 4.69 (s,1H), 4.59 (br. t., 1H, J=6.0 Hz), 4.56 (br. s, 2H), 3.79 (d, 1H, J=6.6Hz), 3.61 (s, 3H) 1.86 (m, 1H), 1.55 (m, 1H), 1.20 (m, 1H), 0.65 (m,1H). HRMS (ESI MS m/z) calculated for C₁₉H₂₁ClN₅O₄ ⁺ (M+H)⁺, 418.1277.found, 418.1277. HPLC RT 16.0 min (100%) in solvent system A, 11.0 min(98%) in system B.

(1′R,2′R,3′S,4′R,5′S)-4′-[2-Chloro-6-(trans-2-phenylcyclopropylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(13b)

Yield 52%. ¹H NMR (CD₃OD): 8.16 (very br. s., 1H), 7.0-7.48 (m, 5H),4.79 (s, 1H), 4.68 (br. s, 2H), 3.88 (d, 1H, J=5.7 Hz), 2.17 (m, 1H)1.97 (m, 1H), 1.65 (m, 1H), 1.29 (m, 2H), 0.74 (m, 1H). HRMS (ESI MSm/z) calculated for C₂₀H₂₁ClN₅O₂ ⁺ (M+H)⁺, 398.1378. found, 398.1372.HPLC RT 20.3 min (99%) in solvent system A, 15.6 min (98%) in system B.

Example 2

This Example illustrates the ability of the compounds in accordance withan embodiment of the invention to bind to A₃ adenosine receptors. Thebinding affinity values are set forth in Table 1.

Receptor Binding and Functional Assays

[¹²⁵I]N⁶-(4-Amino-3-iodobenzyl)adenosine-5′-N-methyluronamide(I-AB-MECA; 2000 Ci/mmol), [³H]cyclic AMP (40 Ci/mmol), and otherradioligands were purchased from Perkin-Elmer Life and AnalyticalScience (Boston, Mass.). [³H]CCPA (2-chloro-N⁶-cyclopentyladenosine) wasa custom synthesis product (Perkin Elmer). Test compounds were preparedas 5 mM stock solutions in DMSO and stored frozen.

Cell culture and membrane preparation: CHO (Chinese hamster ovary) cellsexpressing the recombinant human A₃AR were cultured in DMEM (Dulbecco'smodified Eagle's medium) supplemented with 10% fetal bovine serum, 100units/mL penicillin, 100 μg/mL streptomycin, 2 μmol/mL glutamine and 800μg/mL geneticin. The CHO cells expressing rat A₃ARs were cultured inDMEM and F12 (1:1). Cells were harvested by trypsinization. Afterhomogenization and suspension, cell membranes were centrifuged at 500 gfor 10 min, and the pellet was re-suspended in 50 mM Tris.HCl buffer (pH8.0) containing 10 mM MgCl₂, 1 mM EDTA and 0.1 mg/mL CHAPS(3[(3-cholamidopropyl)dimethylammonio]-propanesulfonic acid). Thesuspension was homogenized with an electric homogenizer for 10 sec, andwas then re-centrifuged at 20,000 g for 20 min at 4° C. The resultantpellets were resuspended in buffer in the presence of adenosinedeaminase (3 Units/mL), and the suspension was stored at −80° C. untilthe binding experiments. The protein concentration was measured usingthe Bradford assay. Bradford, M. M. Anal. Biochem. 1976, 72, 248.

Binding assays at the A₁ and A_(2A) receptors: For binding to human A₁receptors, see (a) Schwabe, U.; Trost, T. Naunyn-Schmiedeberg's Arch.Pharmacol. 1980, 313, 179. (b) Perreira, M.; Jiang, J. K.; Klutz, A. M.;Gao, Z. G.; Shainberg, A.; Lu, C.; Thomas, C. J.; Jacobson, K A. J. Med.Chem. 2005, 48, 4910.

[³H]R-PIA (N⁶-[(R)-phenylisopropyl]adenosine, 2 nM) or [³H]CCPA (0.5 nM)was incubated with membranes (40 μg/tube) from CHO cells stablyexpressing human A₁ receptors at 25° C. for 60 min in 50 mM Tris.HClbuffer (pH 7.4; MgCl₂, 10 mM) and increasing concentrations of the testligand in a total assay volume of 200 μl. Nonspecific binding wasdetermined using 10 μM of CPA (N⁶-cyclopentyladenosine). For humanA_(2A) receptor binding (Jarvis, M. F.; Schutz, R.; Hutchison, A. J.;Do, E.; Sills, M. A.; Williams, M. J. Pharmacol. Exp. Ther. 1989, 251,888-893) membranes (20 μg/tube) from HEK-293 cells stably expressinghuman A_(2A) receptors were incubated with [³H]CGS21680(2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamido-adenosine,15 nM) and increasing concentrations of the test ligand at 25° C. for 60min in 200 μL 50 mM Tris.HCl, pH 7.4, containing 10 mM MgCl₂. NECA (10μM) was used to define nonspecific binding. The reaction was terminatedby filtration with GF/B filters.

Binding assay at the human A₃ receptor: For the competitive bindingassay, each tube contained 50 μL membrane suspension (20 μg protein), 25μL of [¹²⁵I]I-AB-MECA (1.0 nM), Olah, M. E., Gallo-Rodriguez, C.,Jacobson, K. A., Stiles, G. L. Mol. Pharmacol. 1994, 45, 978, and 25 μLof increasing concentrations of the test ligands in Tris.HCl buffer (50mM, pH 8.0) containing 10 mM MgCl₂, 1 mM EDTA. Nonspecific binding wasdetermined using 10 μM of C1-IB-MECA in the buffer. The mixtures wereincubated at 37° C. for 60 min. Binding reactions were terminated byfiltration through Whatman GF/B filters under reduced pressure using aMT-24 cell harvester (Brandell, Gaithersburgh, Md., USA). Filters werewashed three times with 9 mL ice-cold buffer. Radioactivity wasdetermined in a Beckman 5500B γ-counter. IC₅₀ values were converted toK_(i) values as described in Cheng, Y.; Prusoff, W. H. Biochem.Pharmacol. 1973, 22, 3099.

Cyclic AMP accumulation assay: Intracellular cyclic AMP levels weremeasured with a competitive protein binding method. Nordstedt, C.;Fredholm, B. B. Anal. Biochem. 1990, 189, 231; Post, S. R.; Ostrom, R.S.; Insel, P. A. Methods Mol. Biol. 2000, 126, 363. CHO cells thatexpressed the recombinant human or rat A₃AR or the human A₁ or A_(2B)ARwere harvested by trypsinization. After centrifugation and resuspendedin medium, cells were planted in 24-well plates in 1.0 mL medium. After24 h, the medium was removed and cells were washed three times with 1 mLDMEM, containing 50 mM HEPES, pH 7.4. Cells were then treated with theagonist NECA and/or test compound (e.g. 7b) in the presence of rolipram(10 μM) and adenosine deaminase (3 units/mL). After 45 min forskolin (10μM) was added to the medium, and incubation was continued for anadditional 15 mM. The reaction was terminated by removing thesupernatant, and cells were lysed upon the addition of 200 μL of 0.1 Mice-cold HCl. The cell lysate was resuspended and stored at −20° C. Fordetermination of cyclic AMP production, protein kinase A (PKA) wasincubated with [³H]cyclic AMP (2 nM) in K₂HPO₄/EDTA buffer (K₂HPO₄, 150mM; EDTA, 10 mM), 20 μL of the cell lysate, and 30 μL 0.1 M HCl or 50 μLof cyclic AMP solution (0-16 pmol/200 μL for standard curve). Boundradioactivity was separated by rapid filtration through Whatman GF/Cfilters and washed once with cold buffer. Bound radioactivity wasmeasured by liquid scintillation spectrometry.

[³⁵S]GTPγS binding assay: [³⁵S]GTPγS binding was measured by a variationof the method described. (a) Lorenzen, A.; Lang H. Schwabe U. Biochem.Pharmacol. 1998, 56, 1287. (b) Jacobson, K. A.; Ji, X.-d.; Li, A. H.;Melman, N.; Siddiqui, M. A.; Shin, K. J.; Marquez, V. E.; Ravi, R. G. J.Med. Chem. 2000, 43, 2196. Each assay tube consisted of 200 μL buffercontaining 50 mM Tris HCl (pH 7.4), 1 mM EDTA, 1 mM MgCl₂, 1 μM GDP, 1mM dithiothreitol, 100 mM NaCl, 3 U/ml ADA, 0.2 nM [³⁵S]GTPγS, 0.004%3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), and 0.5%bovine serum albumin. Incubations were started upon addition of themembrane suspension (CHO cells stably expressing either the native humanA₁AR or A₃AR, 5 μg protein/tube) to the test tubes, and they werecarried out in duplicate for 30 min at 25° C. The reaction was stoppedby rapid filtration through WHATMAN™ GF/B filters, pre-soaked in 50 mMTris HCl, 5 mM MgCl₂ (pH 7.4) containing 0.02% CHAPS. The filters werewashed twice with 3 mL of the same buffer, and retained radioactivitywas measured using liquid scintillation counting. Non-specific bindingof [³⁵S]GTPγS was measured in the presence of 10 μM unlabelled GTPγS.None of the compounds >10% stimulation; thus, they are antagonists ofthe A₃ adenosine receptor.

TABLE 1 Affinity data for compounds in accordance with an embodiment ofthe invention. In Formula I, R² = Cl, R³ % and R⁴ = OH and R⁵ = HAffinity (K_(i), nM) or % inhibition^(a) Efficacy^(b) Compound R¹ A₁A_(2A) A₃ A₃  7b 3-I-Phenyl-CH₂ 3040 ± 610  1080 ± 310 1.44 ± 0.60 1.0 ±3.2  8b 3-Cl-Phenyl-CH₂ 3070 ± 1500 4510 ± 910 1.06 ± 0.36 2.9 ± 3.7  9b3-Br-Phenyl-CH₂ 1760 ± 1010 1600 ± 480 0.73 ± 0.30 5.8 ± 0.8 10b1-Naphthyl-CH₂ 1120 ± 640  1530 ± 350 1.42 ± 0.12 3.1 ± 0.3 11b2,5-diMeO—Ph—CH₂ 3000 ± 1260 2620 ± 730 1.58 ± 0.56 4.6 ± 3.8 12b2-OH-5-MeO—Ph—CH₂ 1110 ± 300   6870 ± 1440 4.06 ± 0.35 0.4 ± 1.3 13btrans-2-Ph- 1790 ± 1430 2010 ± 890 1.30 ± 0.39 9.7 ± 4.1 cyclopropyl^(a)All experiments were done on CHO or HEK (A_(2A) only) cells stablyexpressing one of four subtypes of human ARs. The binding affinity forA₁, A_(2A) and A₃ARs was expressed as K_(i) values (n = 3-5) and wasdetermined by using agonist radioligands ([³H]CCPA or ([³H] R-PIA),([³H]CGS21680), [¹²⁵I]I-AB-MECA, respectively. The potency at theA_(2B)AR was expressed as EC₅₀ values and was determined by stimulationof cyclic AMP production in AR-transfected CHO cells. A percent inparentheses refers to inhibition of radioligand binding at 10 μM.^(b)measured by [³⁵S]GTPγS binding assay.

In accordance with one method of biological assay, compounds 7b-9b(3-halobenzyl) in the (N)-methanocarba series were potent A₃ ARantagonists with binding K_(i) values of 0.7-1.4 nM. Compound 9b(3-bromobenzyl analogue) proved to be the most potent A₃AR antagonist ofthis series in binding with a K_(i) value of 0.73 nM, and it displayedhigh selectivity (2400-fold and 2190-fold in comparison to the A₁ andA_(2A)AR, respectively). The most A₃AR selective compound was the3-chloro analogue 8b with 2900-fold and 4250-fold selectivity incomparison to the A₁ and A_(2A)AR, respectively. The SAR of substitutionof the N⁶-benzyl group further showed that dimethoxy substitution (11b),fusion of the phenyl ring to a second ring (10b), and extension by onecarbon (i.e., in the rotationally constrained 2-phenylcyclopropylanalogue, 13b) were all tolerated with nanomolar binding affinity at theA₃AR. Compound 12b, a demethylated analogue of 11b, was slightly lesspotent in binding to the A₃AR.

In a functional assay of [³⁵S]GTPγS binding induced by A₃AR activation,7b completely inhibited stimulation by 1 μM NECA(5′-N-ethylcarboxamidoadenosine) with an IC₅₀ of 29.8 nM (FIG. 1).Schild analysis of the right shifts by 7b of the response curves in theinhibition of adenylate cyclase by NECA provided a K_(B) value of 8.9nM.

When compared in the ability to stimulate the A₃AR using multiplefunctional criteria, different results were obtained. In the cAMPassays, compounds 7b and 9b exhibited partial agonism at A₃AR withpercent relative efficacies of 44±6 and 46±4, respectively, and the EC₅₀values were respectively, 12±1 and 4.21±0.6 nM.

Example 3

This example illustrates a method of preparing a radioiodinated compoundin accordance with an embodiment of the invention. Compound 7b having¹²⁵I was prepared as follows. The (radio)iodination of compound 7b onits N⁶-3-iodobenzyl substituent was accomplished in high yield byiododestannylation of a 3-(trimethylstannyl)benzyl precursor through a“cold” iodination reaction as shown in FIG. 6.

Materials and Instrumentation.

Hexamethyltin and other reagents, including pharmacological agents, werepurchased from Sigma-Aldrich Chemical Company, except where noted.Sodium [¹²⁵I]iodide (17.4 Ci/mg) in NaOH (1.0×10⁻⁵ M) was supplied byPerkin-Elmer Life and Analytical Science. ¹H NMR spectra were obtainedwith a Varian Gemini 300 spectrometer using CDCl₃ and CD₃OD as solvents.Chemical shifts are expressed in δ values (ppm) with tetramethylsilane(δ□ 0.00) for CDCl₃ and water (δ 3.30) for CD₃OD. TLC analysis wascarried out on aluminum sheets precoated with silica gel F₂₅₄ (0.2 mm)from Aldrich. HPLC mobile phases consisted of CH₃CN/tetrabutyl ammoniumphosphate (5 mM) from 20/80 to 60/40 in 20 min, flow rate 1.0 ml/min.High-resolution mass measurements were performed on MICROMASS™/WATERS™LCT Premier Electrospray Time of Flight (TOP) mass spectrometer coupledwith a Waters HPLC system.

Preparation of 23:(1′R,2′R,3′S,4′R,5′S)-4′-[2-Chloro-6-(3-trimethylstannylbenzylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(1)

7b (8.95 mg, 0.018 mmol), PdCl₂(PPh₃)₂ (2.7 mg), and hexamethyltin (11μL, 0.054 mmol) were mixed together in anhydrous dioxane (2 ml), and theresulting reaction mixture was stirred at 70° C. for 2 h. The mixturewas concentrated under reduced pressure. The product was purified byflash chromatography by using CHCl₃: MeOH (10:1) as the eluant to affordthe stannyl derivative 23 (9.3 mg, 90%) as an oil. ¹H NMR (300 MHz,CDCl₃), 7.81 (s, 1H), 7.53 (s, 11-1), 7.34 (m, 2H), 7.33 (m, 1H), 6.49(br s, 1H), 4.88 (br s, 2H), 4.00 (m, 2H), 3.71 (s, 1H), 3.65 (m, 1H),3.47 (m, 1H), 2.02 (m, 1H), 1.96 (s, 1H), 1.64 (m, 1H), 1.28 (m, 2H),0.81 (m, 1H), 0.29 (s, 9H). HRMS (M+1)⁺: calculated for C₂₁H₂₇ClIN₅O₂Sn⁺(M+H)⁺535.6338. found 536.0823 HPLC: Rt=22.1 min. HPLC system: 5 mMTBAP/CH₃CN from 80/20 to 60/40 in 25 min, then isocratic for 2 min; flowrate of 1 ml/min.

The trimethylstannyl intermediate 23 (0.1 mg) was reacted sodium [¹²⁵I]iodide in NaOH (1.0×10⁻⁵ M) to obtain [¹²⁵I] 7b, following the proceduredisclosed in Vaidyanathan G., et al., Nat. Protocols 1: 707-713 (2006).

FIG. 7A depicts the non-specific, specific, and total binding of [¹²⁵I]7b on mouse A₃ adenosine receptor. FIG. 7B depicts the extent ofspecific binding as a function of the concentration of the compound. Thecompound was an agonist of the mouse A₃ adenosine receptor.

Example 4

This example illustrates a method of preparing a radiolabeled ligand,that is ⁷⁶Br-labeled compound 9b in accordance with an embodiment of theinvention. Bromine-76 was prepared from an arsenic metal target usingthe ⁷⁵As (³He, 2n) yielding ⁷⁶Br nuclear reaction. The ⁷⁶Br wasprocessed after allowing for the decay of the simultaneously producedBr-75 (t_(1/2)=1.6 h).

An aliquot of the aqueous solution of Br-76 (about 10-20 μl, 18.5-37.0MBq) is added to a 1-mL, reaction vial and the solvent evaporated withargon flow. Trimethylstannyl intermediate 23 in acetonitrile is added tothe vial containing the Br-76 radioactivity and followed by adding 37%peracetic acid in acetonitrile. The vial is sealed and placed on an 80°C. heating block and heated for 30 min. At the end of the reaction, thereaction mixture is loaded onto a PHENOMENEX™ LUNA™ C18 (2) column(250×4.6 mm) and eluted with 100 mM ammonium acetate/acetonitrile(60/40) at a flow rate of 1.2 mL/min. The radioactivity peak containingthe desired product (t_(R)=10 min) is collected and analyzed on aseparate HPLC system for determination of purity and specific activity.

In vivo biodistribution of compound Br-76 labeled compound 9b wascarried out in rats. All studies in live animals were conducted underprotocol approved by the NM Animal Care and Use Committee. Thebiodistribution was evaluated after intravenous administration to adultSprague-Dawley rats. The animals were sacrificed at 15, 30, 60, and 120min and various tissues were harvested for gamma counting. The data arereported in units of percentage of injected dose per gram in FIG. 8. Thecompound exhibited antagonistic properties to the A₃ adenosine receptoralbeit at a low magnitude of uptake. The low uptake may be due to thelower age of the animals. The uptake in the A₃AR-containing testescontinued to increase with time after injection (0.09% ID/g at 15 min to0.18% ID/g at 2 h). Blood continued to provide an input function over 2h. In spite of a potential testes-blood barrier, uptake of theantagonist increased with time, which indicates that the compound may bea viable molecular imaging probe for pathological conditions withelevated A₃AR.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A compound of Formula I:

wherein R¹ is selected from the group consisting of C₃-C₈ cycloalkylC₁-C₆ alkyl, C₃-C₈ dicycloalkyl C₁-C₆ alkyl, C₇-C₁₂ bicycloalkyl C₁-C₆alkyl, C₇-C₁₄ tricycloalkyl C₁-C₆ alkyl, C₆-C₁₄ aryl, C₆-C₁₄ aryl C₁-C₆alkyl, C₆-C₁₄ diaryl C₁-C₆ alkyl, C₆-C₁₄ aryl C₁-C₆ alkoxy, C₁-C₆ alkylcarbonyl, sulfonyl, C₁-C₆ alkyl sulfonyl, C₆-C₁₄ aryl sulfonyl,heterocyclyl C₁-C₆ alkyl, heterocyclyl, heteroaryl C₁-C₆ alkyl,4-[[[4-[[[(2-amino C₁-C₆ alkyl)amino]-carbonyl]-C₁-C₆alkyl]anilino]carbonyl]C₁-C₆ alkyl]C₆-C₁₄ aryl, and C₆-C₁₄ aryl C₃-C₈cycloalkyl, wherein the aryl or heterocyclyl portion of R¹ is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, amino, hydroxyl, carboxy, C₁-C₆ alkoxycarbonyl,C₁-C₆ alkylaminocarbonyl, C₁-C₆ dialkyl aminocarbonyl, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, C₆-C₁₄ aryloxy, hydroxyC₁-C₆ alkyl, hydroxy C₂-C₆ alkenyl, hydroxy C₂-C₆ alkynyl, carboxy C₁-C₆alkyl, carboxy C₂-C₆ alkenyl, carboxy C₂-C₆ alkynyl, aminocarbonyl C₁-C₆alkyl, aminocarbonyl C₂-C₆ alkenyl, aminocarbonyl C₂-C₆ alkynyl, andC≡C—(CH₂)_(n)—COR⁷ wherein R⁷ is selected from the group consisting ofOH, OR⁸, and NR⁹R¹⁰, wherein R⁸ is selected from the group consisting ofC₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl C₁-C₆ alkyl, C₃-C₈dicycloalkyl C₁-C₆ alkyl, C₇-C₁₂ bicycloalkyl C₁-C₆ alkyl, C₇-C₁₄tricycloalkyl C₁-C₆ alkyl, C₆-C₁₄ aryl, C₆-C₁₄ aryl C₁-C₆ alkyl, C₆-C₁₄diaryl C₁-C₆ alkyl; and R⁹ and R¹⁰ are independently selected from thegroup consisting of hydrogen, C₁-C₆ alkyl, and (CH₂)_(n)R¹¹ wherein R¹¹is NR¹²R¹³, wherein R¹² and R¹³ are independently selected from thegroup consisting of hydrogen, C₁-C₆ alkyl, and COR¹⁴ wherein R¹⁴ ishydrogen or C₁-C₆ alkyl; wherein n is an integer from 1 to 10; and thecycloalkyl portion of R¹ is optionally substituted with one or moresubstituents selected from the group consisting of halo, amino, C₁-C₆alkyl, C₁-C₆ alkoxy, C₆-C₁₄ aryloxy, C₁-C₆ hydroxyalkyl, C₂-C₆hydroxyalkenyl, C₂-C₆ hydroxy alkynyl, aminocarbonyl C₁-C₆ alkoxy, andC₆-C₁₄ aryl C₁-C₆ alkoxy; R² is selected from the group consisting ofhydrogen, halo, amino, hydrazido, mercapto, C₁-C₂₀ alkylamino, C₆-C₁₄aryl amino, C₆-C₁₄ aryloxy, C₁-C₂₀ alkyl, C₁-C₂₀ alkoxy, C₁-C₂₀alkylthio, pyridylthio, C₇-C₁₂ cycloalkyl C₁-C₂₀ alkyl, C₇-C₁₂bicycloalkyl C₁-C₂₀ alkyl, C₇-C₁₂ bicycloalkenyl C₁-C₂₀ alkyl, C₆-C₁₄aryl C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₇-C₁₂ cycloalkyl C₂-C₂₀ alkenyl,C₇-C₁₂ bicycloalkyl C₂-C₂₀ alkenyl, C₇-C₁₂ bicycloalkenyl C₂-C₂₀alkenyl, C₆-C₁₄ aryl C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, carboxy alkylC₂-C₂₀ alkynyl, —C≡C—(CH₂)_(m)—C(═O)—O—C₁-C₆ alkyl,—C≡C—(CH₂)_(m)—C(═O)—NH—(CH₂)_(n)—NH₂, —C≡C—(CH₂)_(m)—C₁-C₆ alkyl,—C≡C—(CH₂)_(m)-aryl, wherein m and n are independently 1 to 10, C₇-C₁₂cycloalkyl C₂-C₂₀ alkynyl, C₇-C₁₂ bicycloalkyl C₂-C₂₀ alkynyl, C₇-C₁₂bicycloalkenyl C₂-C₂₀ alkynyl, C₆-C₁₄ aryl C₂-C₂₀ alkynyl, and thealkyl, cycloalkyl, or aryl portion of R² is optionally substituted withone or more substituents selected from the group consisting of halo,hydroxyl, amino, alkylamino, dialkylamino, carboxy, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkyl aminocarbonyl, aminoalkylaminocarbonyl, and trialkylsilyl; R³ and R⁴ are independently selectedfrom the group consisting of hydroxyl, amino, mercapto, ureido, C₁-C₆alkyl carbonylamino, hydroxy C₁-C₆ alkyl, and hydrazinyl; and R⁵ isselected from the group consisting of hydrogen, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, heteroaryl, and C₁-C₆ aminoalkyl; or apharmaceutically acceptable salt thereof.
 2. The compound of claim 1,wherein R¹ is selected from the group consisting of C₆-C₁₄ aryl C₁-C₆alkyl, heteroaryl, and C₆-C₁₄ aryl C₃-C₈ cycloalkyl, wherein the aryl orheteroaryl portion of R¹ is optionally substituted with one or moresubstituents selected from the group consisting of halo, amino,hydroxyl, carboxy, C₁-C₆ alkoxycarbonyl, C₁-C₆ alkylaminocarbonyl, C₁-C₆dialkyl aminocarbonyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxy, C₆-C₁₄ aryloxy, hydroxy C₁-C₆ alkyl, hydroxy C₂-C₆ alkenyl,hydroxy C₂-C₆ alkynyl, carboxy C₁-C₆ alkyl, carboxy C₂-C₆ alkenyl,carboxy C₂-C₆ alkynyl, aminocarbonyl C₁-C₆ alkyl, aminocarbonyl C₂-C₆alkenyl, aminocarbonyl C₂-C₆ alkynyl, and C≡C—(CH₂)_(n)—COR⁷ wherein R⁷is selected from the group consisting of OH, OR⁸, and NR⁹R¹⁰, wherein R⁸is selected from the group consisting of C₁-C₆ alkyl, C₃-C₈ cycloalkyl,C₃-C₈ cycloalkyl C₁-C₆ alkyl, C₃-C₈ dicycloalkyl C₁-C₆ alkyl, C₇-C₁₂bicycloalkyl C₁-C₆ alkyl, C₂-C₁₄ tricycloalkyl C₁-C₆ alkyl, C₆-C₁₄ aryl,C₆-C₁₄ aryl C₁-C₆ alkyl, C₆-C₁₄ diaryl C₁-C₆ alkyl; and R⁹ and R¹⁰ areindependently selected from the group consisting of hydrogen, C₁-C₆alkyl, and (CH₂)_(n)R¹¹ wherein R¹¹ is NR¹²R¹³, wherein R¹² and R¹³ areindependently selected from the group consisting of hydrogen, C₁-C₆alkyl, and COR¹⁴ wherein R¹⁴ is hydrogen or C₁-C₆ alkyl; wherein n is aninteger from 1 to 10; and the cycloalkyl portion of R¹ is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, amino, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₆-C₁₄ aryloxy,hydroxy C₁-C₆ alkyl, hydroxy C₂-C₆ alkenyl, hydroxy C₂-C₆ alkynyl,aminocarbonyl C₁-C₆ alkoxy, and C₆-C₁₄ aryl C₁-C₆ alkoxy, or apharmaceutically acceptable salt thereof.
 3. The compound of claim 1,wherein R¹ is selected from the group consisting of benzyl, phenylcyclopropyl, or 1-naphthyl methyl, wherein the phenyl or naphthylportion of R¹ is optionally substituted with one or more substituentsselected from the group consisting of halo, amino, hydroxyl, carboxy,alkoxycarbonyl, alkylaminocarbonyl, dialkyl aminocarbonyl, C₁-C₆ alkyl,C₁-C₆ alkoxy, phenoxy, hydroxy C₁-C₆ alkyl, hydroxy C₂-C₆ alkenyl, andhydroxy C₂-C₆ alkynyl, or a pharmaceutically acceptable salt thereof. 4.The compound of claim 3, wherein R¹ is benzyl, phenyl cyclopropyl, or1-naphthyl methyl, wherein the phenyl or naphthyl portion of R¹ isoptionally substituted with one or more substituents selected from thegroup consisting of halo, hydroxyl, and alkoxy, or a pharmaceuticallyacceptable salt thereof.
 5. The compound of claim 4, wherein R¹ isbenzyl, or a pharmaceutically acceptable salt thereof.
 6. The compoundof claim 4, wherein R¹ is benzyl substituted with one or moresubstituents selected from the group consisting of halo and C₁-C₆alkoxy, or a pharmaceutically acceptable salt thereof.
 7. The compoundof claim 6, wherein the halo is chloro, bromo, or iodo, or apharmaceutically acceptable salt thereof.
 8. The compound of claim 1,wherein R¹ is selected from the group consisting of 3-chlorobenzyl,3-bromobenzyl, 3-iodobenzyl, 2-hydroxy-5-methoxy-benzyl, and2,5-dimethoxybenzyl, or a pharmaceutically acceptable salt thereof. 9.The compound of claim 4, wherein the phenyl cyclopropyl istrans-2-phenyl-1-cyclopropyl, or a pharmaceutically acceptable saltthereof.
 10. The compound of claim 1, wherein R² is halo, or apharmaceutically acceptable salt thereof.
 11. The compound of claim 10,wherein R² is chloro, bromo, or iodo, or a pharmaceutically acceptablesalt thereof.
 12. The compound of claim 1, wherein R² is—C≡C—(CH₂)_(m)—CH₃, —C≡C—(CH₂)_(m)-aryl, —C≡C—(CH₂)_(m)—C(═O)—O—CH₃,—C≡C—(CH₂)_(m)—C(═O)—NH—(CH₂)_(n)—NH₂, wherein in and n areindependently 1 to 10, wherein the CH₃ or aryl is optionally substitutedwith one or more substituents selected from the group consisting ofhalo, hydroxyl, amino, alkylamino, dialkylamino, carboxy,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, aminoalkyl aminocarbonyl, and trialkylsilyl; or apharmaceutically acceptable salt thereof.
 13. The compound of claim 1,wherein R³ and R⁴ are hydroxyl, or a pharmaceutically acceptable saltthereof.
 14. The compound of claim 1, wherein R⁵ is hydrogen, or apharmaceutically acceptable salt thereof.
 15. The compound of claim 1,wherein R² is chloro, R¹ is 3-chlorobenzyl, 3-iodobenzyl, 3-bromobenzyl,1-naphthylmethyl, 2,5-dimethoxybenzyl, 2-hydroxy-5-methoxybenzyl, ortrans-2-phenyl-cyclopropyl, R³ and R⁴ are hydroxyl, and R⁵ is hydrogen,or a pharmaceutically acceptable salt thereof.
 16. A pharmaceuticalcomposition comprising a compound of claim 1, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier. 17.A radiolabeled compound of Formula I:

wherein R¹ is selected from the group consisting of C₆-C₁₄ aryl, C₆-C₁₄aryl C₁-C₆ alkyl, C₆-C₁₄ diaryl C₁-C₆ alkyl, C₆-C₁₄ aryl C₁-C₆ alkoxy,C₆-C₁₄ aryl sulfonyl, heterocyclyl C₁-C₆ alkyl, heterocyclyl, heteroarylC₁-C₆ alkyl, 4-[[[4-[[[(2-amino C₁-C₆ alkyl)amino]-carbonyl]-C₁-C₆alkyl]anilino]carbonyl]C₁-C₆ alkyl]C₆-C₁₄ aryl, and C₆-C₁₄ aryl C₃-C₈cycloalkyl, wherein the aryl or heterocyclyl portion of R¹ issubstituted with one or more halogen atoms that are radioactive; R² isselected from the group consisting of hydrogen, halo, amino, hydrazido,mercapto, C₁-C₂₀ alkylamino, C₆-C₁₄ aryl amino, C₆-C₁₄ aryloxy, C₁-C₂₀alkyl, C₁-C₂₀ alkoxy, C₁-C₂₀ alkylthio, pyridylthio, C₇-C₁₂ cycloalkylC₁-C₂₀ alkyl, C₇-C₁₂ bicycloalkyl C₁-C₂₀ alkyl, C₇-C₁₂ bicycloalkenylC₁-C₂₀ alkyl, C₆-C₁₄ aryl C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₇-C₁₂cycloalkyl C₂-C₂₀ alkenyl, C₇-C₁₂ bicycloalkyl C₂-C₂₀ alkenyl, C₇-C₁₂bicycloalkenyl C₂-C₂₀ alkenyl, C₆-C₁₄ aryl C₂-C₂₀ alkenyl, C₂-C₂₀alkynyl, carboxy alkyl C₂-C₂₀ alkynyl, —C≡C—(CH₂)_(m)—C(═O)—O—C₁-C₆alkyl, —C≡C—(CH₂)_(m)—(═O)—NH—(CH₂)_(n)—NH₂, —C≡C—(CH₂)_(m)—C₁-C₆-alkyl,—C≡C—(CH₂)_(m)-aryl, wherein m and n are independently 1 to 10, C₇-C₁₂cycloalkyl C₂-C₂₀ alkynyl, C₇-C₁₂ bicycloalkyl C₂-C₂₀ alkynyl, C₇-C₁₂bicycloalkenyl C₂-C₂₀ alkynyl, C₆-C₁₄ aryl C₂-C₂₀ alkynyl, and thealkyl, cycloalkyl, or aryl portion of R² is optionally substituted withone or more substituents selected from the group consisting of halo,hydroxyl, amino, alkylamino, dialkylamino, carboxy, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkyl aminocarbonyl, aminoalkylaminocarbonyl, and trialkylsilyl; R³ and R⁴ are independently selectedfrom the group consisting of hydroxyl, amino, mercapto, ureido, C₁-C₆alkyl carbonylamino, hydroxy C₁-C₆ alkyl, and hydrazinyl; and R⁵ isselected from the group consisting of hydrogen, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, heteroaryl, and C₁-C₆ aminoalkyl; or apharmaceutically acceptable salt thereof.
 18. The radiolabeled compoundor salt of claim 17, wherein the halogen atom of R¹ is ¹⁸F, ⁷⁶Br, or¹²⁵I.
 19. The radiolabeled compound or salt of claim 18, wherein R¹ is3-bromobenzyl or 3-iodobenzyl, R² is halo, R³ and R⁴ are hydroxyl, andR⁵ is hydrogen.